Benoit Fatou (Presenter)
PRISM Laboratory, University of Lille
Bio: I am a postdoctoral fellow at the PRISM Laboratory and I am working on new technological innovation for cancer research. I did my PhD in the same laboratory and my project was to make a proof of concept about the development of a new instrument able to perform real-time analysis by mass spectrometry for guided surgery but also for dermatology and cosmetology.
Authorship: Benoit FATOU (1), Philippe SAUDEMONT (1), Eric LEBLANC1, Denis VINATIER (1), Maxence WISZTORSKI (1), Cristian FOCSA (2), Michel SALZET (1), Michael ZISKIND (2), Isabelle FOURNIER (1)
(1) Laboratoire PRISM, INSERM U1192, Université Lille 1, Villeneuve d’Ascq, FRANCE (2) Laboratoire PhLAM, UMR 8523, Université Lille 1, Villeneuve d’Ascq, FRANCE
| Short Abstract To date, detection of early diagnosis is an important issue for clinical studies. Nevertheless, highly specific sets of pathology related compounds are generally found in tissues and only few of them will get to body fluids. There is a need for new tools allowing for in vivo diagnosis with minimal invasiveness. Ambient mass spectrometry techniques are becoming some powerful tools permitting to generate specific molecular profiles on tissue without any sample preparation and have shown to be efficient for diagnostic purpose. Here, we want to explore the clinical potential of a new instrument able to perform sampling using laser ablation and analyze the ablated particles by mass spectrometry in real-time conditions. The instrument setup and some results about the clinical application will be presented, especially on the ovarian cancer. |
Long Abstract
Characterization of biological tissues in clinical problematics related to solid cancer is currently performed by two strategies. The first consists to localize an abnormal volume using imaging techniques such as Magnetic Resonance Imaging (MRI). The second strategy is to sample biopsies of this volume and the pathologist realizes an ex vivo diagnosis at a cellular level based on histological staining and molecular techniques using targeted biomarkers such as immunohistochemistry. These strategies have some limitations especially the localization of tumor region inside the abnormal volume and the use of targeted biomarkers not specific of a unique cancer. The challenge is thus to get a set of biomarkers using untargeted strategies and to go as closely as possible to the tumor environment. In the context of clinical research, mass spectrometry (MS) has shown its capacities to be an untargeted technique for the molecular characterization for ex vivo in situ analysis of tissues of biopsies, in particular mass spectrometry imaging (MSI) which can also keep the molecular localization. It has been evidenced that molecular signatures generated in situ from tissues by MS or MSI are correlated with pathological grade or stage. Since a decade, MS heads to ambient surface analysis by performing the sampling at atmospheric pressure and room temperature without sample preparation. This ambient surface analysis is realized by ambient ion sources which can perform desorption and ionization of the molecules in one or two steps. However, all of these techniques require the use of several biopsies which can increase significantly the time of diagnosis and the risks of inflammation.
For clinical research, the challenge is to obtain the diagnosis based on MS inside the operating rooms to have a real-time MS analysis during the sampling and there are some requirements for example the conservation of physiological functions, the low-depth and painful sampling and the biocompatibility of the instrument. The aim is thus to develop a MS-based instrument which is able to perform a remote sampling from the inlet of the MS instrument. It has been recently shown that the iKnife, developed by Prof. Z. Takats, is an alternative solution for in vivo medical conditions (1). This instrument is based on the collection of smokes emitted by an electrical scalpel while cutting tissues, called Rapid Evaporative Ionization Mass Spectrometry (REIMS). Then, the smokes are aspirated by a tube, inserted inside the scalpel and connected to the inlet of the MS instrument. Several publications described this technology and evaluated the clinical potential on different studies of cancer. Despite the fact that this is the first in vivo MS-based diagnostic instrument, some constraints are observed especially the delayed healing because of the use of electric scalpel and the ablation depth is dependent on the surgeons.
Here, we want to develop another technical solution allowing to achieve diagnosis in a less invasive manner using laser ablation (2). The advantages of laser ablation are the tunability due to the large wavelength range, the precise control of the fluence which corresponds to the laser energy per surface unit, and the transport of the laser energy without contact with the tissue so no contamination could be observed between two sampling points. This instrument is based in situ sampling using a nanosecond pulsed laser tuned at 2940 nm. This wavelength is chosen to target the endogenous water molecules for desorption and ionization of the biomolecules. The laser beam was brought onto the sample surface by coated mirrors and a convergent lens. To perform real-time analysis, the ablated material is aspirated through a Teflon tubing connected to the mass spectrometer and assisted through a Venturi pump.
These experiments showed the real-time conditions by observing the evolution of the generated signal. Indeed, the signal increases directly after the beginning of the laser irradiation and it decreases less than 3 seconds after the laser turned off. The real-time MS acquisition during the irradiation time shows some intact charged compounds assigned as lipids and metabolites using lipid databases. To verify the nature of the observed signals, selection of precursor ions to obtain MS/MS spectra was performed using our instrument and we are able to identify the most abundant ions which correspond to the phospholipid species.
Then, we evaluated the clinical potential of our instrument by an oncology study on ovarian cancer. The distinction of benign and tumor biopsies is observed in real-time MS analysis. To confirm the differences of molecular profiles, some biopsies were sampled from 90 patients and these biopsies were analyzed and the MS spectra were recorded. The raw files were then exported and statistical analyses were realized. A classification model is created based on the use of an algorithm called support vector machine (SVM) and were are able to observe that around 90% of the data set were good classified. The correlation between our molecular data and the pathologist was possible in the same way that the improvement of the diagnosis given by the pathologist.
To demonstrate the application for in vivo conditions, we tested the system on human skin (fingers) from healthy individuals and we are able to show some distinct molecular profiles according to the gender. The obtained results show the feasibility of the technology as well as its low-depth sampling (about 5µm per laser shot) which demonstrate its clear interest for clinical applications. This new instrument could be a powerful contribution for the surgeons to obtain the diagnosis using MS directly in operating rooms during the surgery procedure.
References & Acknowledgements:
References:
(1)Balog J, Szaniszlo T, Schaefer K-C, et al. Identification of biological tissues by rapid evaporative ionization mass spectrometry. Analytical Chemistry. 2010;82(17):7343-50
(2)Fatou B, Saudemont P, Leblanc E, Vinatier D, Mesdag V, Wisztorski M, Focsa C, Salzet M, Ziskind M, Fournier. In vivo Real-Time Mass Spectrometry for Guided Surgery Application. Scientific Reports. 2016 May 18;6:25919. doi: 10.1038/srep25919.
Acknowledgments
Supported by grants from University Lille 1 (BQR to Dr. M. Ziskind and PhD grant for B. Fatou), Ministère de l’Enseignement Supérieur et de la Recherche via Institut Universitaire de France (Prof. I. Fournier) and SIRIC ONCOLille (Prof. I. Founier) Grant INCa-DGOS-Inserm 6041, INSERM PhysiCancer (project SPIDERMASS, Prof. M. Salzet), SATT Nord de France (Project SPIDERMASS, Prof. I. Fournier), ANR Santé Bien-être (Project Reality’MS, Prof. I. Fournier).
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